Heinz Sep2009 Effect of high-pressure processing on Listeria spp. and on the textural and microstructural properties of cold smoked salmon
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Heinz Sep2009 Effect of high-pressure processing on Listeria spp. and on the textural and microstructural properties of cold smoked salmon

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Investigation of the effect of high-pressure processing (HPP) at very short time on the inactivation of Listeria innocua was conducted as well as the effect on texture and microstructure. Lipid ...

Investigation of the effect of high-pressure processing (HPP) at very short time on the inactivation of Listeria innocua was conducted as well as the effect on texture and microstructure. Lipid oxidation, colour
and background bacterial flora were studied as well. HPP at 700–900 MPa for 10 s increased the inactivation of L. innocua in cold smoked salmon from 4500 cfu/g to nondetectable level (<0.3 cfu/g). L. innocua was more sensitive to HPP than the background flora tested. The product presented good microbiological quality and there was no indication of lipid oxidation. The effect of HPP on the redness of the product was not observed, however immediate effect on the lightness was noticed and the salmon
becomes lighter in colour as a function of both time and pressure. The effects on the microstructure increased with both time and pressure and were most significant at 900 MPa and 60 s. The effect on microstructure coincides with the reduction of the bacteria. The knowledge from this study provides
information for the industry on the development of HPP at 400–900 MPa with short pressure time of less than 60 s.

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Heinz Sep2009 Effect of high-pressure processing on Listeria spp. and on the textural and microstructural properties of cold smoked salmon Heinz Sep2009 Effect of high-pressure processing on Listeria spp. and on the textural and microstructural properties of cold smoked salmon Document Transcript

  • ARTICLE IN PRESS LWT - Food Science and Technology xxx (2009) 1–9 Contents lists available at ScienceDirect LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt Effect of high-pressure processing on Listeria spp. and on the textural and microstructural properties of cold smoked salmon Birna Gudbjornsdottir a, *, Asbjorn Jonsson b, Hannes Hafsteinsson b, Volker Heinz c a Bollagarðar 33, 170 Seltjarnarnes, Iceland b ´ Matis ohf, Skulagata 4, 101 Reykjavik, Iceland c ¨ German Institute of Food Technology (DIL e.V.), Professor-von-Klitzing-Straße 7, D-49610 Quakenbruck, Germany a r t i c l e i n f o a b s t r a c t Article history: Investigation of the effect of high-pressure processing (HPP) at very short time on the inactivation of Received 6 January 2008 Listeria innocua was conducted as well as the effect on texture and microstructure. Lipid oxidation, colour Received in revised form and background bacterial flora were studied as well. HPP at 700–900 MPa for 10 s increased the inac- 5 August 2009 tivation of L. innocua in cold smoked salmon from 4500 cfu/g to nondetectable level (<0.3 cfu/g). Accepted 21 August 2009 L. innocua was more sensitive to HPP than the background flora tested. The product presented good microbiological quality and there was no indication of lipid oxidation. The effect of HPP on the redness of Keywords: the product was not observed, however immediate effect on the lightness was noticed and the salmon Smoked salmon Microstructure becomes lighter in colour as a function of both time and pressure. The effects on the microstructure High pressure increased with both time and pressure and were most significant at 900 MPa and 60 s. The effect on Listeria microstructure coincides with the reduction of the bacteria. The knowledge from this study provides Shelf life information for the industry on the development of HPP at 400–900 MPa with short pressure time of less than 60 s. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction & Gibbs, 2006; Yoon, Burnette, Abou-Zied, & Whiting, 2004). However these methods could not prevent the growth of this Global aquaculture production has increased in recent years and bacterium. If the organism cannot be eliminated and growth- is now growing more rapidly than all other animal food production inhibiting steps are not introduced, the hazard has to be controlled sectors. A great proportion of farmed Atlantic salmon reaches the by limiting shelf life (at 4  C) to ensure that no more than 100 cells/ worldwide market as a cold smoked product, but smoking is one of g are present at the time of consumption. Time limits for storage the oldest processing methods that have been used to extend the may need to be established by each processor because it should shelf life of food. While the world production increases, problems reflect the initial level of the organism in freshly produced product. related to bacterial contamination in cold smoked salmon still To meet demands from consumer about safe food with prolonged persist (Gombas, Chen, Clavero, & Scott, 2003; Gram, 2001; Gud- shelf life, it is necessary to develop a new processing method for mundsdottir et al., 2005). Due to the fact that the temperature smoked salmon. Over the last decade, investigators have explored during the smoking never exceeds 28  C it does not have any the possibilities of applying some novel technologies against this significant effect on Listeria spp. Many processing parameters have pathogen. High-pressure processing (HPP) is one of these prom- been tested to minimise the occurrence of Listeria monocytogenes, ising technologies. It is a non-thermal preservation technique that such as combinations of NaCl and low temperature, other preser- depends on pressure, time, temperature and product characteris- vatives (e.g. diacetate and lactate) or by the use of bioprotective tics and it allows micro-organisms to be inactivated with fewer cultures that can inhibit growth at refrigerated temperatures changes in texture, colour and flavour as compared to conventional (Fonnesbech Vogel, Yin, Hyldig, Mohr, & Gram, 2006; Huss, technologies (Carpi, Gola, Maggi, Rovere, & Buzzoni, 1995; Cheftel, ´ Jørgensen, & Fonnesbech Vogel, 2000; Gram, 2001; Tome, Teixeira, 1995; Knorr, 1993; Torres & Velazquez, 2005). High-pressure processing (HPP) of foods was first reported by Hite (1899) who used this technology to increase the shelf life of * Corresponding author. Tel.: þ354 5611257, þ354 8684258 (mobile). milk. Since then several studies on different food items have been E-mail address: birna.gudbjornsdottir@gmail.com (B. Gudbjornsdottir). published. Most of the studies related to the application of HPP to 0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2009.08.015 Please cite this article in press as: Gudbjornsdottir, B., et al., Effect of high-pressure processing on Listeria spp. and on the..., LWT - Food Science and Technology (2009), doi:10.1016/j.lwt.2009.08.015
  • ARTICLE IN PRESS 2 B. Gudbjornsdottir et al. / LWT - Food Science and Technology xxx (2009) 1–9 seafood have been conducted on its effect on proteins (Ang- 3–4 kg each was randomly selected from a large population that supanich, Edde, & Ledward, 1999), muscle colour (Amanatidou was slaughtered that day and kept on ice. Two days later the salmon et al., 2000; Ohshima, Ushio, & Koizumi, 1993), lipids (Chevalier, was filleted and cold smoked at Reykofninn smokehouse, which is Bail, & Ghoul, 2001; Ohshima, Nakagawa, & Koizumi, 1992) and located in the area of Reykjavik. The fillets were salted in brine, bacteria (Amanatidou et al., 2000; Smelt, 1998). The effect of HPP containing 8 g NaCl in 100 ml of water for 24 h, and smoked at 24 h on L. monocytogenes has been intensively studied regarding the at 18–20  C. Each fillet was vacuum packed and delivered imme- effect of treatment time (Patterson, Quinn, Simpson, & Gilmour, diately to the Icelandic Fisheries Laboratory (IFL). The following day 1995; Simpson & Gilmour, 1997), of pressure (Shigehisa, Ohmori, the fillets were cut into 30–50 g pieces (4 cm  6 cm) and vacuum Saito, Taji, & Hayashi, 1991) and the minimum conditions of the packed by Magic VacTM Champion. Before the packaging, one half three parameters (pressure, time, temperature) to maximize the of the samples were contaminated (spiked) with 1 ml of reduction of cell viability (Ritz et al., 2000). Lakshmanan and Dal- 2  105 cfu/ml of bacterial suspension (L. innocua, E-34, the most gaard (2004) showed that pressure at 250 MPa did not inactivate L. resistance bacteria from the pre-study) to obtain a final concen- monocytogenes but lag phases of 17 and 10 days were observed at 5 tration of 103–104 cfu/g in the salmon sample. Dilution was made in and 10  C, respectively. Pressure at 200 MPa had a marked effect on a Butterfields buffer. The other half were not contaminated, but both the colour and the texture of chilled cold smoked salmon. high-pressure processed and used for textural and microstructural Another study showed that high-pressure treatment of salmon analysis. The day after (day 5 from slaughter) the samples were paste extended shelf life from 60 to 180 days at 3 or 8  C without transported to Berlin University of Technology, Department of Food significant chemical, microbiological, or sensory changes and Biotechnology and Food Process Engineering. On day 6 and 7 the completely inactivated pathogens present in the inoculated sample smoked salmon samples were processed with high-pressure at (Carpi et al., 1995). Montero, Gomez-Estaca, and Gomez-Guillen 400, 500, 600, 700, 800 and 900 MPa for 10, 20, 30 and 60 s. On day (2007) presented that cold smoked dolphinfish processed under 8 the samples were transported back to Iceland where they were severe salting and smoking conditions (2.93% salt and 82 ppm examined for microbiology at IFL and texture and microstructure at phenol) in combination with pressurization at 300 MPa at 20  C for the Technical Institute of Iceland (IceTec). The temperature during 15 min kept L. monocytogenes counts under the detection limit this transportation was documented with a logger and the average throughout 100 days of storage. The resistance of micro-organisms temperature was 4  C (data not shown). Water content, water to pressure varies considerably and mainly depends on pressure, activity, lipid (fat) content, pH, NaCl content and Thiobarbituric acid time and temperature. By increasing the pressure and time of (TBARS) value were determined to characterize the material used treatment the number of L. monocytogenes in hard cheese, meat for this study. Samples for these analyses were taken from the raw products and fruit juice decreased proportionally (Fonberg-Broczek material and at different steps throughout the process. et al., 2005). L. monocytogenes has been shown to be very sensitive to pressure changes and due to the cost of HPP it would be desirable to increase treatment pressure and keep the treatment time short 2.3. Water -, fat- and salt content, lipid oxidation, water activity (Chen, Guan, & Hoover, 2006). Most of the studies related to and pH analysis application of HPP of seafood apply 200–700 MPa pressure for 3, 5, 10, 15 or 20 min (Torres & Velazquez, 2005). Water content was measured according to ISO 6496 (1999). The However, new development in high-pressure technology enables sample was heated in an oven at 103  C Æ 2  C for 4 h. Water high pressure to be reached in 10 s. The objective of this research was content corresponds to the weight loss. to study the effect of HPP (400–900 MPa) on the survival of Listeria Total fat was determined by extraction with petroleum ether, innocua and the characteristics (microstructure, texture and colour) boiling range 40–60  C using an extraction apparatus 2050 Soxtec of cold smoked salmon during 10, 20, 30 and 60 s. The changes in Avanti Automatic System (AOCS, 1998). counts of total viable psychrotrophic bacteria (TVC), lactic acid Salt content was measured according to AOAC (2000). Soluble bacteria (LAB) and Bacillus spores (PCA) were investigated. chloride was extracted from the sample with water containing nitric acid. The chloride content of the solution was titrated with 2. Materials and methods silver nitrate and the end point was determined potentiometrically. Lipid oxidation. Thiobarbituric acid reactive substances (TBARSs) 2.1. Preparation of bacterial culture were determined by a modified version (Sørensen & Jørgensen, 1996) of the extraction method described by Vyncke (1970, 1975) In order to decide which strain should be selected for this study with few modifications. The sample size was reduced to 15 g and a pre-study was performed on six strains of L. monocytogenes and homogenized with 30 ml of 7.5 g/100 g of trichloroacetic acid 2 strains of L. innocua. All strains were obtained from IFL-collection solution containing 0.1 g/100 g of both propyl gallate and EDTA. The after isolation from product or processing environment during cold absorbance of samples and standards were measured at 530 nm. smoking. The strains were cultivated overnight in Tryptic Soy Broth TBARS, expressed as mmol malondialdehyde per kilogram of sample supplemented with 0.6 g Yeast Extract/100 g (Difco) at 35  C and (mmol MDA/kg), was calculated using malondialdehyd-bis-(diethyl subcultured twice. All these strains have been compared by acetate) as standard. a genetic typing technique using pulsed-field gel electrophoresis Water activity was measured by using Aw – Wert – Messer (Gudmundsdottir et al., 2005). The following strains were tested for (Durotherm) capsule at 22  C and kept in an incubator for at least the efficacy of HPP on reducing the bacterial number: L. innocua, 4 h before a reading was taken. Calibration and temperature strains EU2173/E-34 and EU2172/E-33; L. monocytogenes, strains E1, corrections were made according to the manufacturer’s E5, H-01-170-2, L-327, L-435 and L-462. instructions. pH analysis. Approximately 5 g of minced tissue was mixed with 2.2. Raw material and sample preparation the same amount of water (weight), and pH measurement was made using the PHM 80 Portable Radiometer Analytical Copenha- 2.2.1. Cold smoked salmon (CSS) gen, with an immersed electrode according to instructions of the Atlantic salmon (Salmo salar) was farmed and slaughtered at manufacturer’s manual. Rifos hf. on the north coast of Iceland. A sample of 50 fishes of All analysis was done in duplicate or triplicate. Please cite this article in press as: Gudbjornsdottir, B., et al., Effect of high-pressure processing on Listeria spp. and on the..., LWT - Food Science and Technology (2009), doi:10.1016/j.lwt.2009.08.015
  • ARTICLE IN PRESS B. Gudbjornsdottir et al. / LWT - Food Science and Technology xxx (2009) 1–9 3 2.4. Microbial analysis 2.6. Storage trials with spiked samples of cold smoked salmon For the microbial analysis 25 g of the salmon samples were The storage trial consisted of samples after two HPP treatments added to 225 ml of Maximum Recovery Diluent (MRD, Difco) and (500 and 900 MPa) and of untreated samples used as a control. The mixed in stomacher (Stomacher 400, A. J. Seward, London, UK) storage temperature was 5.5  C. The samples were tested for for 2 min. Two additional 10-fold dilutions were prepared by microbial changes (TVC, LAB and Bacillus spores), the survival of L. adding 1 ml of the previous dilution to 9 ml of MRD. Total viable innocua and for lipid oxidation on days 5, 12, 26 and 41. psychrotrophic count (TVC) was determined by spread plating on Modified Long and Hammer (LH) agar according to Van Spreek- 2.7. Microstucture ens (1974) with 1 g NaCl/100 g (15  C for 5–7 days). Lactic acid bacteria (LAB) were determined by spread plating on Nitrite Samples for microstructure analysis were collected from the Actidione Polymyxin (NAP) agar for 22  C for 5 days (Modified HPP treated salmon pieces using a cork knife, 11 mm in diameter. from: Davidson & Cronin, 1973). To confirm existence of LAB, They were embedded in plastic tubes 15 mm in diameter, 30 mm catalase test was performed. The most probable number method length (Kartell, Novigilo, Italy) containing optical coherence (MPN) was used to enumerate Listeria and the media used and tomographical (OCT) compound (embedding medium) (Tissue Tek, steps were according to USDA–FSIS Method (2002). Listeria Sakura, Torrance, CA) and frozen in liquid nitrogen. Freezing (below enrichment broth base (UVM formulation, Oxoid) was used as the À80  C) occurred in approximately 40 s. The frozen specimens pre-enrichment step followed by inoculation to Fraser broth were stored at À80  C until cryosectioning and staining. The (Oxoid) with a subcultured to a plate of Modified Oxford Agar specimens were sectioned frozen at À27  C in a Leica CM1800 (Difco) from all black tubes. In this case the 3-tube 3 dilution cryostat (Leica, Heidelberg, Germany) for transverse cuts, 10 mm (3 Â 3) format was used. The detection limit for this method is thick. Sections were mounted on glass slides and stained using MPN 0.3 cfu/g or 1 cfu/100 ml (solid or liquid sample). One mil- Orange G (0.5 g CI 16230 (Polysciences, Warrington, PA), 99.0 ml lilitre of the homogenated sample was transferred into the first of distilled water, 1.0 ml acetic acid). The sections were washed with three test tubes, each containing 10 ml of UVM in double distilled water and stained for 5 min in methyl blue solution (0.07 g concentration. The next three consecutive dilutions were made in CI 42780 (Sigma, St. Louis, MO), 99.0 ml water and 1.0 ml acetic single concentration of UVM. This allowed us to achieve sampling acid). The stained samples were washed for 5 min with distilled sizes of 1, 0.1 and 0.01 g of salmon per 10 ml. For Bacillus spore water, dried at room temperature and mounted with MOUNTEX count, 10 ml of the 1/10 mixture were heated at 75  C for 30 min. ¨ (Histolab, Goeteburg, Sweden). The samples were examined in an The pour plate technique was done on Plate Count Agar (Difco). optical microscope, Leica DM RA2 at 100Â magnification and Plates were incubated at 35  C for 2 days. All analyses were done images captured using a Leica DC300F digital camera mounted on in duplicate. the microscope. The pictures of the microstructure of samples where analysed in Leica QWin software, where the amount of 2.5. High-pressure processing (HPP) noncellular material was measured. In other words, the spaces between cells were analysed. 2.5.1. Efficacy of HPP on reducing Listeria spp. (pre-study) The pre-study of HPP was carried out at IceTec using an Auto- 2.8. Texture measurements clave Engineering (Erie, PA, USA) high-pressure system. Maximum design pressure for the system was 500 MPa at room temperature. Warner–Bratzler shearing blade with a thickness of 3.21 mm, The size of the sample holder was 2.4 l. Pressure transmitting length of 125 mm and width of 70 mm was assembled to the medium was 5 ml/100 ml oil (Kutwell 42 from Exxon) in water TA.XT2Ò Texture Analyser (Surrey, England). It shears or cuts solution. Cell suspensions (1 ml) inoculated to a gauze were placed through the sample with a test speed of 2.5 mm/s. The computer in a plastic bag and vacuum packed by Magic VacTM Champion. The software was set to plot a force versus time plot and the results bacterial strains were high-pressure treated at 350 MPa for 5 and were expressed as the maximum peak force (shear force in N) 20 min at 22  C. A total of 5 min were needed to reach 350 MPa required to shear through the sample. Other parameter calculated whereas pressure decompression took 30 s. was the area under the curve which describes the total amount of work required to cut through the sample. This test method incor- 2.5.2. High-pressure processing of smoked salmon porates compression of fibres beneath the blade, tension in the High-pressure processing of smoked salmon was carried out at adjoining fibres and shearing of the fibres (Bouton, Harris, & The Berlin University of Technology, Department of Food Biotech- Shorthouse, 1975). Textural measurements were performed on nology and Food Process Engineering, Berlin, Germany, in a designed samples from the fillet. Each sample was measured three times. and constructed lab-scale high-pressure system (High Pressure Research Center, Unipress Equipment Division, Sokolowska 29/37, 2.9. Colour analysis Warsaw, Poland). Maximum design pressure for the system was 1000 MPa at an operating temperature range of À25–100  C. The The intensity of the flesh colour was measured by using the volume of the sample holder was 0.75 l. A mixture of water and MiniScan XE plus from HunterLab using the D65 light source. The glycol (propylene glycol 1.2 propanediol; 50:50) was used as instrument records the L* (lightness À intensity of white colour), a* a pressure transmitting medium. The most HPP-resistance strains (redness À intensity of red colour) and b* (yellowness À intensity of (L. innocua (E-34)) from the pre-study were chosen to continue the yellow colour) values. Each sample was measured in triplicates. main study. Spiked and unspiked vacuum packed salmon samples were high-pressure processed at 400, 500, 600, 700, 800 and 2.10. Statistical analysis 900 MPa pressure for 10, 20, 30 and 60 s. Temperature during holding time (after adiabatic heating) did reach 42  C in all trials. Analysis of variance (ANOVA) was carried out on microbial data Unpressurized samples were used as a control. 10 s were needed to and TBARS value in the statistical program NCSS 2000 (NCSS, Utah, reach 400, 500 and 600 MPa and 20 s to 25 s to reach 700, 800 and USA) to compare the effect of different treatment. The program 900 MPa. calculates multiple comparisons using Tukey–Kramer Multiple- Please cite this article in press as: Gudbjornsdottir, B., et al., Effect of high-pressure processing on Listeria spp. and on the..., LWT - Food Science and Technology (2009), doi:10.1016/j.lwt.2009.08.015
  • ARTICLE IN PRESS 4 B. Gudbjornsdottir et al. / LWT - Food Science and Technology xxx (2009) 1–9 Comparison Test. The same test was used for the texture and Table 1 microstructure, using statistical program Sigmastat version 2.03 Survival of L. innocua in HPP treated cold smoked salmon. Most Probable Number (MPN). Samples measured two days after high-pressure treatment. Average of two (Systat Software Inc., California, USA). Results were considered experiments. significant at p < 0.05. High pressure (MPa) cfu/g (MPN) 10 s 20 s 30 s 60 s 3. Results Spiked 400 MPa >110 >110 >110 >110 Spiked 500 MPa >110 >110 >110 110 Spiked 600 MPa >110 16.7 1.9 0.3 3.1. Characteristics of cold smoked salmon Spiked 700 MPa 0.8 <0.3 0.3 <0.3 Spiked 800 MPa 0.3 <0.3 <0.3 <0.3 The results for dry matter, salt content, fat content and TBARS Spiked 900 MPa <0.3 <0.3 <0.3 <0.3 were presented as mean of two or three values (mean Æ SD). The Unspiked 500 MPa <0.3 <0.3 cold smoked salmon contained 37.2 g/100 g Æ 0.4 dry matter and Unspiked 900 MPa <0.3 <0.3 3.2 g NaCl/100 g Æ 0.1 corresponding to 4.9% Æ 0.2% water phase salt (WPS). The fat content was 9.6 g/100 g Æ 0.4 and pH was 6.1 Æ 0.1. The TBARS value was low, e.g. 3.9 mmol/kg Æ 0.1 mmol/kg. 3.3. Storage trials with spiked samples of cold smoked salmon TVC on LH and LAB count on NAP was determined in cold smoked salmon at different processing steps, in the raw filet, after 3.3.1. Microbial analysis salting and washing and after the smoking step (Fig. 1). The The initial bacterial counts in the samples are described in chapter bacterial count increased from <1 log cfu/g to 2.6 log Æ 0.2 log cfu/ 3.1. No Listeria spp. was detected in unspiked-unpressurized control g and to 2.7 log Æ 0.1 log cfu/g, respectively. The increase of TVC samples, not even after 41 days storage at 5.5  C (Figs. 2 and 3), occurred after the salting and washing step while count of LAB indicating a good quality and safety of the raw material used in this increased steadily during the whole process and in the final product experiment. Fig. 2 shows that L. innocua was reduced significantly LAB were the dominant flora of the total count. It is well known that (p < 0.05) after 60 s at 500 MPa. One log reduction was observed and most of LAB will grow on LH and therefore it is stated that LAB is the number decreased during storage. No reduction was observed dominant of the spoilage flora in the samples. Neither Listeria nor after 10 s and it remained unchanged until after more than 26 days. Bacillus spores were detected in any of these samples. Neither Lis- By increasing the treatment time to 20 and 30 s it appeared that the teria nor Bacillus spores were detected in any of these samples. bacteria were shocked in some way as the total number decreased during the first 12 days of storage but after that the bacteria recov- ered and the number increased. No L. innocua was detected 5 days 3.2. Challenge test with L. innocua after HPP treatment at 900 MPa (Fig. 3) but after storage at 5.5  C for 26–41 days low level (0.3–20 cfu/g) was detected. It is difficult to say The number of L. innocua in overnight culture was if this indicates that some of the cells survived and could recover after 2.03 Â 109 cfu/ml. Cold smoked salmon was spiked with 104 dilu- some storage time or this difference is due to individual difference tion of overnight culture and the final number of L. innocua in the between samples. Tables 2 and 3 depicts changes in the TVC and LAB spiked samples was 4.5 Â 103 cfu/g. Increasing the pressure from counts in the vacuum-packed cold smoked salmon after pressure 400 MPa to 900 MPa had a significant effect on the reduction, from treatment and during storage at 5.5  C. The LAB count was consid- >110 cfu/g to <0.3 cfu/g after 10 s (Table 1). At 400–500 MPa the erably higher than TVC or log 5.5 compared to log 3.7 in untreated reduction of bacteria was less than 1–1.5 log cycle. By increasing the samples indicating that by using LH at 15  C some LAB cannot grow pressurization time at 600 and 700 MPa the reduction increased. (Table 2). Unfortunately the LAB were not identified down to species. These results indicate that the pressure needs to be up to At day 5 the TVC count was 0.2–1.2 log lower in samples treated at 700–900 MPa to be efficient enough to reduce the number of 500 MPa compared to untreated samples (Table 2). During the L. innocua in cold smoked vacuum packed salmon with regards to storage the number increased steadily and after the 41 days storage the safety of the product. The initial number of the bacterium in the about 0.8–2.7 log increases were observed. The number of LAB spiked product tested was rather high, around 4500 cfu/g, which is reduced significantly (p < 0.05) compared to untreated samples but not the real situation on the market. during storage 4–6 log increase was observed indicating that the LAB 4 5 4 Log number/g 3 Log Number/g 3 2 2 1 1 0 0 5d 12 d 26d 41d Raw fillet Fillet after salting Cold smoked Storage time, days and washing salmon Fig. 2. Growth of Listeria innocua (MPN) during storage of cold smoked salmon at Fig. 1. Distribution of bacteria during processing of cold smoked salmon. Total viable 5.5  C. Cold smoked salmon had been treated with 500 MPa for 10 (-,-), 20 (-Â-), 30 psychrotrophic counts (TVC) determined by spread plating on LH (Long and Hammer (-A-) and 60 (---)s. Samples not treated with HPP used as control (unspiked->-, -) and lactic acid bacteria (LAB) on NAP (Nitrite Actidione Polymyxin ,). Mean of two spiked-:-). Mean of two samples (mean Æ SD). Listeria concentration before pressure samples (mean Æ SD). was 4.5 Â 103 cfu/g. Please cite this article in press as: Gudbjornsdottir, B., et al., Effect of high-pressure processing on Listeria spp. and on the..., LWT - Food Science and Technology (2009), doi:10.1016/j.lwt.2009.08.015
  • ARTICLE IN PRESS B. Gudbjornsdottir et al. / LWT - Food Science and Technology xxx (2009) 1–9 5 5 12 4 10 Log number/g 8 TBA (µm ol / kg) 3 6 2 4 1 2 0 0 5d 12 d 26d 41d Fresh Control 500 700 900 500 700 900 500 700 900 Storage time, days sample MPa- MPa- MPa- MPa- MPa- MPa- MPa- MPa- MPa- 10s 10s 10s 30s 30s 30s 60s 60s 60s Fig. 3. Growth of Listeria innocua (MPN) during storage of cold smoked salmon at 5.5  C. Cold smoked salmon had been treated with 900 MPa for for 10 (-,-), 20 (-Â-), Fig. 4. Changes in TBARS value as an indicator for rancidness during storage at 5.5  C 30 (-A-) and 60 (---)s. Samples not treated with HPP used as control (unspiked->-, for 18 (black column) and 41 (blank column) days after HPP treatment at 500, 700 and spiked-:-). Mean of two samples (mean Æ SD). Listeria concentration before pressure 900 MPa for 10, 30 and 60 s Mean of two samples (mean Æ SD). was 4.5 Â 103 cfu/g. can recover more quickly maybe because of the lack of competition from background flora. After HPP treatment at 900 MPa LAB were can recover quickly after HPP treatment at 500 MPa. When the clearly so damaged that they could not recover even after some samples were treated at 900 MPa pressure a significant (p < 0.05) storage time as shown in Table 3. Bacillus spores were not detected in reduction of TVC of log 2–3 was observed when compared to the any samples. nonpressure treated samples on day five. The reduction was 2.2 log after 10 s, 2.7 log after 20 s, 1.6 log after 30 s and 1.9 log after 60 s 3.3.2. Lipid oxidation (Table 3) indicating a better effect when the treatment time was Fig. 4 shows the results from measurements on lipid oxidation. shorter. It should be pointed out that the number increased more The TBARS was low (4.7–7.3 mmol/kg). All the smoked fillet samples rapidly after treatment at 500 MPa time indicating that the cells are showed slightly higher TBARS values than the fresh fillet samples more damage after treatment at 900 MPa. The effects on LAB count (Fig. 4). The difference is not significant (p > 0.05). When treated at were very clear. After treatment at 900 MPa 2.9–4.1 log decrease was 900 MPa for 60 s, then the TBARS value decreased during storage from observed and after storage for 26 days the count was 0.7–2 log lower 7.31 mmol/kg after 18 days to 5.91 mmol/kg after 41 day. This differ- after treatment for 30 and 60 s compared to 10 and 20 s indicating ence is probably due to the individual difference between samples. better effect after longer treating time. The HPP treatment had significant (p < 0.05) effect on the growth of LAB, both at 500 and 900 MPa and it was obvious that the recovery after 900 MPa treat- ments was difficult. A reduction of 4 log was observed after treat- 3.4. Microstructure ment time for 20–60 s at 900 MPa. After storage for 26 days the LAB count was 3.4–6.1 log cfu/g in 500 MPa treated samples compared to With increased pressure and longer processing time the space 8.1 log cfu/g in untreated samples indicating that LAB are sensitive to between the myofibrils (cells) increased (Fig. 5). Shrinkage on HPP treatment. The number of LAB on NAP was up to 1.2–4 log higher myofibrils is negligible at 400 and 500 MPa, for processing time of than total count on LH after treatment at 500 MPa. The difference was 10 and 20 s. At 600 MPa and higher and for processing time of 30 not as high after 900 MPa. The increase of LAB was considerably and 60 s the effects showed a considerable shrinkage of myofibrils faster after HPP treatment at 500 MPa (Table 2) indicating that they resulting in increased space between the myofibrils. Table 2 Microbial evaluation in vacuum packed cold smoked (spiked) after HPP treatment at 500 MPa during storage at 5.5  C. Means of two samples (mean Æ SD). Treatment time (s) Total viable psychrotrophic count on LH (log cfu/g) Lactic acid bacteria on NAP (log cfu/g) 0 10 20 30 60 0 10 20 30 60 Storage time (days) 5 3.68 Æ 1.21 2.86 Æ 0.64 2.52 Æ 0.04 3.49 Æ 0.96 2.80 Æ 0.12 5.55 Æ 0.77 1.45 Æ 0.64 2.45 Æ 0.04 2.69 Æ 0.56 2.18 Æ 0.04 12 3.47 Æ 0.38 2.27 Æ 0.52 2.48 Æ 0.14 na 3.24 Æ 0.80 5.40 Æ 3.03 3.02 Æ 2.01 2.24 Æ 0.14 3.58 Æ 1.8 3.77 Æ 2.81 26 4.71 Æ 0.56 3.65 Æ 0.04 3.22 Æ 0.71 5.28 Æ 1.44 4.27 Æ 0.18 8.14 Æ 0.09 6.10 Æ 0.72 3.42 Æ 1.43 6.09 Æ 1.54 5.38 Æ 0.18 41 4.80 Æ 0.96 4.60 Æ 1.69 5.20 Æ 0.12 5.22 Æ 0.57 3.56 Æ 0.79 8.78 Æ 0.24 7.58 Æ 0.12 6.44 Æ 1.22 7.79 Æ 0.91 6.30 Æ 0.85 Table 3 Microbial evaluation in vacuum packed cold smoked (spiked) after HPP treatment at 900 MPa during storage at 5.5  C. Treatment time (s) Total viable psychrotrophic count on LH (log cfu/g) Lactic acid bacteria on NAP (log cfu/g) 0 10 20 30 60 0 10 20 30 60 Storage time (days) 5 3.68 Æ 1.21 1.45 Æ 0.21 1.00 Æ 0 2.11 Æ 1.57 1.79 Æ 1.12 5.55 Æ 0.77 na 1.30 Æ 0 1.57 Æ 0.81 1.00 Æ 0 12 3.47 Æ 0.38 1.45 Æ 0.21 1.30 Æ 0.43 1.00 Æ 0 1.00 Æ 0 5.40 Æ 3.03 4.52 Æ 1.01 1.15 Æ 0.21 1.39 Æ 0.55 1.00 Æ 0 26 4.71 Æ 0.56 2.29 Æ 0.30 2.99 Æ 0.27 1.69 Æ 0.12 1.45 Æ 0.21 8.14 Æ 0.09 3.11 Æ 0.86 4.00 Æ 0.48 2.00 Æ 0 2.37 Æ 0.52 41 4.80 Æ 0.96 na* na na 1.00 Æ 0 8.78 Æ 0.24 na na na 1.00 Æ 0 * na – no growth detected. Please cite this article in press as: Gudbjornsdottir, B., et al., Effect of high-pressure processing on Listeria spp. and on the..., LWT - Food Science and Technology (2009), doi:10.1016/j.lwt.2009.08.015
  • ARTICLE IN PRESS 6 B. Gudbjornsdottir et al. / LWT - Food Science and Technology xxx (2009) 1–9 Fig. 5. Effect of high pressure on the microstucture of cold smoked salmon. Frozen specimen were prepared two days after treatment and stored at À80  C until examined. 3.5. Texture measurements reason is that the control samples were collected from various part of the fillet, but not from the same location on the fillet. Generally Our results showed non-significant differences in toughness there was a gradual increase in toughness as the pressure was between the control sample and various HPP treatments for 20 s increased. Samples processed at 900 MPa in 10, 30 and 60 s were (Fig. 6). The control sample had a large standard deviation and the significantly tougher than samples processed at 400 MPa (data not Please cite this article in press as: Gudbjornsdottir, B., et al., Effect of high-pressure processing on Listeria spp. and on the..., LWT - Food Science and Technology (2009), doi:10.1016/j.lwt.2009.08.015
  • ARTICLE IN PRESS B. Gudbjornsdottir et al. / LWT - Food Science and Technology xxx (2009) 1–9 7 30 70 25 60 Toughness (N) 20 50 L value 15 40 10 30 5 20 0 10 0 400 500 600 700 800 900 0 Pressure (MPa) 0 400 500 600 700 800 900 Pressure (MPa) Fig. 6. Effect of high-pressure process for 20 s on smoked salmon. Samples measured two days after high-pressure treatment. Mean of three samples (mean Æ SD). Fig. 8. The lightness (L) of the high-pressure processed smoked salmon (20 s) measured by MiniScan from HunterLab. Samples measured two days after high- pressure treatment. Mean of three samples (mean Æ SD). shown). Each sample was measured in triplicates. Similar effects were also observed for 10, 30 and 60 s processing times, for different HPP treatments. delays the lag phase which can consequently prolong the storage time. However, the initial number of the L. innocua in the final 3.6. Colour analysis product tested was rather high or 4500 cfu/g which might not reflect the situation in the industry. Beaufort et al. (2007) and Cortesi, Sarli, The effect of high-pressure processing on lightness in the Santoro, Murru, and Pepe (1997) stated that typical contamination in smoked salmon could be seen visually. The lightness increased naturally contaminated product may be about or below 1 cfu/g. from the control to 900 MPa when processed for 20 s (Fig. 7). The Jørgensen and Huss (1998) reported that initial numbers of L. mon- effects were similar for salmon processed for 10, 30 and 60 s (data ocytogenes in cold smoked salmon were <10 cells/g and only two not shown). The effects are more visible for the interior of the samples (of 32 positive) contained between 103 and 104 cfu/g after salmon than the surface. There was a significant increase in light- 3–7 wk of storage. In this study Listeria spp. was not detected in ness (L* values) in all samples processed with high-pressure naturally contaminated salmon after storage for 41 day at 5.5  C. The treatment compared to the control (Fig. 8). On the contrary, there data presented in this paper indicates that Listeria spp. is more was no effect of the high-pressure process on the redness (a* value) sensitive to HPP than the background flora tested. As already of the smoked salmon (Fig. 9). explained then the results show that LAB became predominant of the spoilage flora in the samples. It have been stated that LAB often 4. Discussion dominates the microbial flora in smoked fish products during refrigerated storage (Magnusson & Traustadottir, 1982). As a result The effects of HPP on micro-organisms in food are of great smoked fish products have a prolonged shelf life, since the Gram- interest and it has been shown that the effect of the HPP treatment negative spoilage flora is somewhat inhibited (Jeppesen & Huss, on L. monocytogenes is depending on processing parameters such as 1993) and that LAB appear to be well adapted in vacuum packages time and pressure (Ritz et al., 2000). Investigation of the effect of and more resistant than Gram-negative bacteria to the high salt both pressure and treatment time on the inactivation of L. innocua content found in smoked salmon products (Hansen, Gill, & Huss, was conducted. The results indicate that the pressure needs to reach 1995). The number of LAB on NAP was up to 1.2–3 log higher than 700–900 MPa to reduce the number of L. innocua to the level where total count on LH after treatment at 500 MPa. The difference was not the safety of the product can be ensured and are not detectable as high after 900 MPa or mainly below 1 log. The difference observed (<0.3 cfu/g) 5 days after the HPP treatment. HPP above 600 MPa on the LH and NAP is relative high in the present study compared to Fig. 7. Photographs of high-pressure processed smoked salmon for 20 s. Photos taken two days after high-pressure treatment. Please cite this article in press as: Gudbjornsdottir, B., et al., Effect of high-pressure processing on Listeria spp. and on the..., LWT - Food Science and Technology (2009), doi:10.1016/j.lwt.2009.08.015
  • ARTICLE IN PRESS 8 B. Gudbjornsdottir et al. / LWT - Food Science and Technology xxx (2009) 1–9 35 900 MPa, the toughness was similar as in the control sample. The probable reason for this is that the measurement of the textural 30 properties was performed on samples, located in different parts of 25 the fillets. Jonsson, Sigurgisladottir, Hafsteinsson, and Kristbergs- son (2001) and Sigurgisladottir, Hafsteinsson, Jonsson, Lie, Nort- a value 20 vedt, Thomassen, and Torrissen (1999) reported the difference in 15 toughness on different location in fresh Atlantic salmon fillets. The 10 study of Jonsson et al. (2001) showed variation in the value of maximum force, to cut through the samples along the fillet of fresh 5 salmon from 15 N near the head and 42 N near the tail. Studies 0 have shown increased tissue firmness in fresh bluefish at high 0 400 500 600 700 800 900 pressure (200 MPa for 10 min) (Ashie & Simpson, 1996; Simpson, Pressure (MPa) 1998). Fig. 9. The redness (a) of the high-pressure processed (20 s) smoked salmon measured Previously it has been shown that the colour of the fish is the by MiniScan from HunterLab. Samples measured two days after high-pressure treat- main quality aspect of interest to consumers both in the German ment. Mean of three (mean Æ SD). and the French market (Sigurgisladottir, Torrisen, Lie, Thomassen, & Hafsteinsson, 1997; Torrissen et al., 2000). Negative effects on the colour, caused by HPP of smoked salmon, could therefore be the literature (Leroi, Joffraud, Chevalier, & Cardinal, 2001). It is a hindering factor regarding the application of this new technology. expected that LAB can grow on LH. It can be speculated that pro- The results of this study show no effect on the redness, but Ama- cessing parameters for smoked products such as dry salting and natidou et al. (2000) had earlier reported a significant reduction of brine injection influence the microbial spoilage (Hansen et al.,1995). redness caused by HPP. HPP has immediate effect on the lightness. In this study the fillets were salted in brine, 8 g NaCl in 100 ml water, Even treatment time of 10 s and pressure of 400 MPa had signifi- for 24 h, and smoked at 24 h at 18–20  C. It can also be assumed that cant effect on the lightness of cold smoked salmon. Lightness then not all species of LAB grow well on LH due to the lack of glucose in increased as a function of treatment time and pressure and coin- this medium, or because of the low incubation temperature (15  C). cides with the visual changes where the salmon became lighter in This difference was also detected in the untreated samples and after colour as a function of both time and pressure. However, due to the five days it was 2 log which is more in accordance with previously short processing time the highest value for lightness never excee- reported results (Leroi et al., 2001) but after 41 days it was 4 log. The ded 62. A threshold value of acceptability for smoked salmon has increase of LAB was considerably faster after HPP treatment at not been reported, but for fresh salmon a threshold value of 70 is 500 MPa, compared to TVC indicating that they can recover more considered as unacceptable (Amanatidou et al., 2000). Studies on quickly, possibly because of the lack of competition from other cod – and mackerel muscle showed that the colour of the muscle background flora. The results indicate that HPP can extend the became lighter with increasing pressure (Ohshima et al., 1993). microbial shelf life of cold smoked salmon when exposed to 900 MPa Because of the cost of HPP it would be desirable to increase treat- to 26 days compared to suggested limited shelf life for a period ment pressure and keep the treatment time as short as possible (14–21 days) where growth of Listeria spp. is unlikely to take place or (Chen et al., 2006). It should be pointed out that the high temper- to reach levels >100 cfu/g (Huss et al., 2000). ature (42  C) during the holding time (after adiabatic heating) does There was no indication of lipid oxidation which is in accor- have an obvious effect on the appearance and on the texture of cold dance with previously reported results (Espe, Nortvedt, Lie, & smoked salmon. Therefore it is important to carry out this tech- Hafsteinsson, 2001; Espe, Nortvedt, Lie, & Hafsteinsson, 2002). nique with cooled condition. Because of high levels of polyunsaturated lipids the salmon is susceptible to deterioration by oxidation that can affect product 5. Conclusion quality of the salmon and affect the sensory quality, but values has not been found to be so high that the fish should be regarded as The present study yielded significant results for cold smoked oxidized (Espe et al., 2002). All the values obtained in this study salmon food processing and revealed that the combination of high were considered low and safe for consumption if compared to pressure and short treatment time is very effective to improve the limits for frozen fish that is 18 mmol/kg according to Robles-Mar- safety of cold smoked products. However further studies are tinez, Cervantes, and Ke (1982). necessary and hurdle effects of some parameters should be evalu- The effect of HPP on the microstructure of cold smoked salmon ated due to the changes in the visual appearance and texture. For was minimal at 400 MPa, but increased with both time and pres- example, further studies on the effect of MAP in combination with sure and is most significant at 900 MPa and 60 s. When Fig. 5 is HPP should be performed. Decreasing the working temperature compared to results in Table 1, where the greatest reduction of L. during HPP treatment will most likely reduce the effect on micro- innocua occurs, it is clear that the effect on microstructure coincide structure and texture. This new development is promising to meet with the reduction of the bacteria. The effect at 500 MPa was not as the requirement for prolonged shelf life of ready-to-eat cold obvious. It is known that HPP affects microstructure of meat and smoked salmon with high microbiological quality and safety. fish at pressure between 200 and 400 MPa, depending on the type (Ledward, 1998). Gudmundsson and Hafsteinsson (2001) have also Acknowledgements reported the effect of HPP at 300 MPa on gaping in salmon fillets. Their results indicated more gaping in salmon fillets, caused by This work was funded by the Icelandic Research Council. Thanks shrinkage of the myofibrils and increased space between the are extended to Icelandic companies Rifos and Reykofninn and to myofibrils. the Technical University of Berlin (TUBER), Department of Food The effect of HPP on the textural properties is not clear. It is Biotechnology and Food Process Engineering for assistance with the likely that toughness decreases when salmon fillets were treated at experiments. Special thanks to Margret Bragadottir, Asa Thorkels- 400 and 500 MPa for 10, 20, 30 and 60 s, compared to the control dottir and the staff of microbiological and chemical lab at IFL and sample. When pressure was increased to 700 MPa and up to IceTech. Please cite this article in press as: Gudbjornsdottir, B., et al., Effect of high-pressure processing on Listeria spp. and on the..., LWT - Food Science and Technology (2009), doi:10.1016/j.lwt.2009.08.015
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